PROJECT SUMMARY Aging is the primary risk factor for cardiovascular disease (CVD), and by 2030, 40% of Americans will have some form of CVD incurring a huge economic toll on society. Despite significant gains in treating CVD over the last few decades, much remains unknown about why cardiovascular dysfunction increases with age. One potential cause of cardiovascular aging is cellular senescence. This complex stress response can be beneficial or detrimental, depending on the physiological context. Two hallmarks of the response are: 1) a permanent arrest of cell proliferation, preventing development of cancer in the face of genotoxic insults; 2) development of a senescence-associated secretory phenotype (SASP) -- the transcriptional upregulation and secretion of numerous inflammatory cytokines, chemokines, growth factors and proteases. Senescent cells increase with age, including in the cardiovascular (CV) system, and the SASP is associated with a variety of age-related pathologies, including cardiac and vascular dysfunction. Senescent cells accumulate with age in the hearts and vasculature of mice and humans, but it is not known if cellular senescence is a cause or consequence of cardiovascular aging. We developed a novel mouse model that permits the visualization and elimination of senescent cells in vivo and their isolation from tissues, making it possible to study the function of integrated systems -- such as the heart and vasculature -- with and without senescent cells. We propose to test the novel hypothesis that senescent cells, and particularly the SASP, are an important mechanistic process driving CV aging. To test this hypothesis, we will develop three specific aims. Aim 1: In vivo consequences of senescence in the CV system. This aim will determine when and where senescent cells arise in the cardiovasculature, using an acute model, as well as naturally aged mice. We will employ our novel mouse model, the 3MR mouse in which we can eliminate cells to contrast CV function with and without senescent cells; Aim 2: Role of senescent cells in modulating cardiac and arterial function. This aim will determine the sensitivity of different cell types in the CV to senescence, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells isolated from both arteries and intact hearts. We will determine how secreted factors from senescent cells influence function of other cell types using co-cultures and genetic strategies; Aim 3: Translational validation of senescent markers identified in Aims 1-2. Having established specific protein and gene expression signatures for different cell types as a result of senescence in aims 1 and 2, in this final aim we will use a novel translational model ? biopsies of endothelial cells from humans to validate our predictions about senescence in different aged humans. Overall our program will provide novel insights into the role of senescence as a major mediator of age- related CVD, and potentially provide new targets of opportunity to combat this devastating disorder.